Macrolactone pyrrolizidine alkaloids (MPAs) are broadly distributed plant metabolites that possess hepatotoxic, carcinogenic, mutagenic, and in some cases, tumor inhibiting properties. Their toxicology has been shown to have human health and economic implications in areas where MPAs enter the food chain. This research is directed toward chemical synthesis of selected MPAs and toward understanding the molecular basis for their physiological action. Specific MPAs targeted for study are swazine, madurensine, doronenine, merenskine N-oxide, neosenkirkine, and rosmarinine. The constituent necic acids (swazinecic, bulgarsenecic, merenskinecic) and necine bases (crotanecine, otonecine, rosmarinecine) will be prepared enantiospecifically from (R) or (S) beta-citronellol in case of necine acids and from (2R)-2-hydroxybutyrolactam in the case of necine bases. Two methods will be examined for regiospecific coupling of the activated necic acid with its appropriate pyrrolizidine base, the latter as a N-protected borane. One of these will employ photolysis of a S-necyl xanthate to generate an intermediate alpha-lactone whereas the other will make use of a Mitsunobu esterification protocol. A new hypothesis for metabolic activation of MPAs via their 1,2-epoxides will be tested by preparing these systems from natural alkaloids and studying their chemical conversion to pyrrolic dehydroMPAs. The metabolic significance of these epoxides as compared with the isomeric MPA N-oxides would be assessed with radiolabeled alkaloids. A postulate that dehydroMPAs, in analogy with mitomycin C, can form covalent, interstrand crosslinks in duplex DNA will be evaluated in a collaborative project with Dr. Paul B. Hopkins, University of Washington. The prediction that the metabolically active, pyrrolic form of the MPA will exhibit 5'dCpG covalent binding with a distinct base sequence preference will be tested in a synthetic, radiolabeled (32P) oligonucleotide duplex. A hydroxyl radical-mediated, sequence-specific DNA cleavage technique that locates crosslinks with single-base resolution will be used in conjunction with fragment analysis by polyacrylamide gel electrophoresis.